The long-range goal of this research is to elucidate the role of cyclic nucleotide- and Ca ions -stimulated proteins phosphorylation in the physiological and pharmacological regulation of smooth muscle contraction. There is increasing evidence that many of the responses of neurotransmitters and hormones are mediated by phosphorylation of key proteins. With regard to smooth muscle, recent studies indicate that: (1) a modulator-dependent protein kinase causes the Ca ions-dependent phosphorylation of the 20,000 Mr myosin light chain; this may be an obligatory link in the regulation of myosin ATPase; (2) Ca ions-uptake by isolated membrane fractions from several smooth muscles is stimulated by cyclic AMP, presumably via increased protein phosphorylation. The methodology for detecting changes in the phosphorylation of specific proteins in contracting myometrium has recently developed in our laboratory. Thus, for the first time in intact smooth muscle, reversible phosphorylation of a 20,000 dalton protein has been associated with spontaneous and hormone-induced contraction. In our present studies, we propose to more clearly define the role of this protein, as well as cyclic nucleotide-sensitive proteins, in the regulation of uterine contraction. Our approach will allow a comparison of specific proteins phosphorylated in intact tissue with those phosphorylated in isolated fractions. We will carefully examine the relationship between the degree of phosphorylation of these proteins and: (1) the amount of contraction and relaxation of ray myometrium; (2) Ca ions sequestration properties of relatively pure fractions of myometrial sarcolemma and sarcoplasmic reticulum. The proposed investigation is important for: (1) resolving the current controversy on the existence of effective cellular compartments for cyclic nucleotides in smooth muscle; (2) elucidating the role of the Ca ions-stimulated phosphorylation in uterine contraction; (3) providing fundamental information on the mechanisms by which neurotransmitters, hormones, and therapeutic agents may regulate smooth muscle tension.